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X-Ray Absorption Spectroscopic Analysis of the High-Spin Ferriheme Site in Substrate-Bound Neuronal Nitric-Oxide Synthase1

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X-Ray Absorption Spectroscopic Analysis of the High-Spin Ferriheme Site in Substrate-Bound Neuronal Nitric-Oxide Synthase1 J. Biochem. 130. 191-198 (2001) X-Ray Absorption Spectroscopic Analysis of the High-Spin Ferriheme Site in Substrate-Bound Neuronal Nitric-Oxide Synthase1 Nathaniel J. Cosper,* Robert A. Scott,*,2 Hiroyuki Hori,•õ Takeshi Nishino,•õ and Toshio Iwasaki•õ,2 Department of Chemistry; University of Georgia, Athens. GA 30602-2556, USA; and *Depatment of Biochemistry and Molecular Biology, Nippon Medical School. 1-1-5 Sendogi. Buukyo-ku, Tokyo 113-8602 Received March 27, 2001; accepted April 17, 2001 Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide through two stepwise oxygenation reactions involving NƒÖ-hydroxy-L-argin ine, an enzyme-bound intermediate. The NƒÖ-hydroxy-L-arginine- and arginine-bound NOS ferriheme centers show distinct, high-spin electron paramagnetic resonance sig nals. Iron X-ray absorption spectroscopy (XAS) has been used to examine the structure of the ferriheme site in the NƒÖ-hydroxy-L-arginine-bound full-length neuronal NOS in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin. Iron XAS shows that the high-spin ferriheme sites in the NƒÖ-hydroxy-L-arginine- and arginine-bound forms are strikingly similar, both being coordinated by the heme and an axial thiolate ligand, with an Fe-S distance of ca. 2.29 A. Cu2+ inhibition slightly affects the spin-state equilibrium, but causes no XAS-detectable changes in the immediate ferriheme coordination environ ment of neuronal NOS. The structure and ligand geometry of the high-spin ferriheme in arginine-bound neuronal NOS are essentially identical to those of the NƒÖ-hydroxy-L-argi nine-bound form and only slightly affected by the divalent cation inhibitor of consitu tive NOS. Key words: electron paramagnetic resonance spectroscopy, heme, neuronal nitric oxide synthase, nitric oxide synthase, X-ray absorption spectroscopy. Nitric oxide (NO) serves as a physiological vasodilator, neu ine, and an interdomain binding site for a tetrathiolate zinc rotransmitter, and cytostatic agent in mammalian cells, center (10-13). During NO synthesis, the two flavins (FAD and is generated by a family of enzymes termed nitric oxide and FMN) in the reductase domain mediate electron trans synthases (NOSs) (1-5). All known NOS isoforms are each fer from NADPH to the heme group in the oxygenase do catalytically active as a homodimeric, bidomain flavo-heme- main, where oxygen binding and activation take place (1, 6, 14-16). Spectroscopic studies on the heme centers of the protein, consisting of a flavin-containing C-terminal reduc tase domain and a home-containing N-terminal oxygenase NOS isoforms involving absorption, electron paramagnetic domain (6-9). The oxygenase domain also contains binding resonance (EPR), resonance Raman, and magnetic circular sites for 6(R)-5,6,7,8-tetrahydro-L-biopterin (H4BP), L-argin- dichroism techniques, along with mutational analysis, have suggested that an endogenous thiolate sulfur donor ligand is coordinated to the central heme iron, as in the cases of 1 This investigation was supported in part by Grants-in-Aid for Sci entific Research on Priority Areas from the Ministry of Education, cytochrome P450 and chloroperoxidase (17-26). Science, Sports and Culture of Japan ("Biometallics" 08249104 to Neuronal NOS (nNOS) generates NO through the two- T.N. and "Biomachinery" 11169237 to T.I.), Grants-in-Aid from the step oxygenation reactions that convert L-arginine to citrul Ministry of Education, Science. Sports and Culture of Japan line. Its activity is dependent on oxygen, NADPH, H4BP, ( 09480167 to T.N., and 8780599 and 11780455 to T.I.), and by the and Ca2+-calmodulin (1, 6, 14). The first step is the forma National Institutes of Health (GM 42025 to R.A.S.). The XAS data tion of NƒÖ-hydroxy-L-arginine (NOHA) as an enzyme-bound were collected at SSRL. which is operated by the Department of En ergy. Division of Chemical Sciences. The SSRL Biotechnology pro intermediate (1, 14, 27). It has been proposed, by analogy to gram is supported by the National Institute of Health. Biomedical P450 mechanisms, that the conversion of L-arginine to Technology Program. Division of Research Resources. NOHA requires reduction of the ferriheme, binding of O2 to 2 To whom correspondence should be addressed: Robert A. Scott, form the ferrous-O2 adduct, and activation of O2 to form a Phone +1-706-542-2726, Fax: +1-706-542-9454, E-mail: rscott@ reactive high-valent oxo-ferryl porphyrin, a ƒÎ-cation radical uga.edu; Toshio Iwasaki, Phone: +81-3-3822-2131 (Ext. 5216), Fax; species which allows oxygen insertion into L-arginine (1, 14, +81-3-5685-3054, E-mail: iwasaki/[email protected] 28). The second step, i.e. conversion of NOHA to citrulline Abbreviations: eNOS, endothelial nitric oxide synthase; EPIC elec tron paramagnetic resonance; EXAFS, extended X-ray absorption and NO, is thought to require the binding of O2 to the fer fine structure; FT, Fourier transform; H4BP, (6R)-5,6.7.8-tetrahydro rous enzyme, and the involvement of the ferric peroxide -L- biopterin; iNOS, inducible nitric oxide synthase; NO. nitric oxide; intermediate and an NO-L-arginine radical to form a puta NOHA, NƒÖ-hydroxy-L-arginine; NOS, nitric oxide synthase; nNOS, tive iron-peroxo-NOHA radical intermediate (1, 14, 28). neuronal nitric oxide synthase; SSRL, Stanford Synchrotron Radia Although a possible two-step oxygenation mechanism for tion Laboratory; XAS, X-ray absorption spectroscopy. NOS has been proposed on the basis of analogy to P450-N- c 2001 by The Japanese Biochemical Society hydroxylation and P450-aromatase systems, there are sub- Vol. 130, No. 2, 2001 191 192 N.J. Cosper et al. tie differences between NOS isoforms and cytochromes of analytical grade. P450, which suggest that the NOS reaction may be more A heterologous expression system for the full-length complex. First, there is little homology in amino acid wild-type nNOS in Escherichia coli was constructed w th sequence or overall protein folding between the NOS oxyge the pCWori+ vector (47-49) and the chaperonin expression nase domain and cytochrome P450, thereby excluding NOS vector pKY206 (pACYC184; Nippon Gene, Toyama) carry- from the cytochrome P450 gene family All NOS isoforms ing the E. coli chaperonin groELS genes (kindly provided are each catalytically active only as a homodimer (1, 6, 8, by Dr. K. Ito, Kyoto University), as reported previously (31). 29), and mutagenesis studies have indicated that appropri The recombinant nNOS produced in E. coli strain BL21 ate cross-talk between the N-terminal hook region and the was purified on ice or at 4•Ž essentially as described in the substrate-binding distal pocket of NOS isoforms is essential literature (49), except that purification was conducted by for proper activation and regulation of the citrulline- and 2•Œ,5•Œ-ADP Sepharose 4B column chromatography (Amer NO-forming activity (12, 30-32). Second, all NOS isoforms sham Pharmacia Biotech), followed by Sephacryl S300HR show an absolute requirement of H4BP for NO synthase and DEAF Sepharose Fast Flow column chromatography activity (33, 34). The exact role of H4BP in NOS catalysis (Amersham Pharmacia Biotech) (31), and that the over- remains controversial. Recent studies have suggested a night dialysis step was omitted. H4BP (30ƒÊM) and L-argi possible redox function for the H4BP cofactor (13, 35, 36), nine (1mM) were supplied during the purification. The but the redox cycling of H,BP in the NOS catalytic cycle catalytic activity and purity of the purified enzyme were seems to be different from that observed with the aromatic comparable to those previously reported for recombinant amino acid hydroxylase reaction, which requires a non nNOS by others (49). heme iron center in close proximity to H4BP (13, 36). NOS activity was measured by monitoring the conver To fully understand the mechanismof NO synthesis,it is sion of [14C-U]-L-Arg to [14C-U]-L-citrulline, as described essential to determine the changes that the catalyticcenter previously (50). The standard assay was performed at 25•Ž undergoes during the reaction cycle(28, 37). The iron coor in an assay mixture containing 16.7mM HEPES-NaOH dination structures of a variety of heme proteins including buffer, pH 7.4, 4.2mM Tris-HCl buffer, pH 7.4, 667ƒÊM cytochromesP450 and chloroperoxidasehave been studied EDTA, 167ƒÊM EGTA, 667 ƒÊM DTT, 16.7ƒÊM [14C-U]-L- by X-ray absorption spectroscopy(XAS) (37-40). The metal Arg, 667ƒÊM NADPH, 1.2mM CaCl, 6.7ƒÊg of calmodulin, ligand bond distancesobtained by XAS and high-resolution 1.25ƒÊM FAD, 1.25ƒÊM FMN, 2.5ƒÊM H,BP and the en X-ray crystallographicanalysis are in close agreement for zyme, in a total volume of 30ƒÊl. The specific activity of [14C-U]-L-Arg several different oxidation and spin states of cytochrome -Are used in the assays was 11.84GBq/mmol. P450 (37). Extensive XAS analyses have been conducted Absorption spectra were recorded with a Hitachi U3210 with cytochromesP450 and chloroperoxidaseto elucidate spectrophotometer or a Beckman DU-7400 spectrophotom the change in the iron coordinationin relation to the cata eter. Electron paramagnetic resonance (EPR) spectra of lytic mechanism and to elucidate the structures of stable several different batches of purified nNOS were measured intermediate species (37-42).Although several X-ray crys at JEOL, Tokyo, with a JEOL JES-TE200 spectrometer tal structures of eNOS and iNOS oxygenatedomains com equipped with an ES-CT470 Heli-Tran cryostat system, in plexed with substrate and inhibitors have been reported which the temperature was monitored with a Scientific recently (10-13, 43), the NOS holoprotein is a complex, Instruments digital temperature indicator/controller Model multidomain flavohemoproteinwhose flavin and heme cen 9650 and the magnetic field was monitored with a JEOL ters are probably positioned near each other (21).
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